In the activation of transcription the GACKIX motif, a small conserved domain plays an important role with two potential docking sites for activators and has been found in CBP and its homolog p300 as well as the unrelated coactivator ARC105. The functions of activator*GACKIX domain complexes are diverse and include cellular growth, hematopoiesis, synaptic plasticity, and lipid homeostasis; thus, dysregulation of these complexes are also linked to many diseases from leukemia to Alzheimer's and Huntington's disease. Chemical tools are sought to dissect the individual functions of activator*GACKIX domain complexes and to provide a potential therapeutic model. However small molecules have yet to be designed that can distinguish between the GACKIX domain of a particular coativator such as p300 versus CBP. In this research plan the site-directed strategy known as Tethering is applied to match orthogonal disulfide-containing fragments with cysteine-modified GACKIX (cysGACKIX) domain. First a liquid chromatography-mass spectrometry Tethering screen was used to identify disulfide fragments that target the MLL-binding site of the cysGACKIX domain. A novel fluorescence polarization Tethering screen streamlined the identification of small molecules inhibitors directed at the c-Myb and CREB- binding site of the cysGACKIX domain. The disulfide fragments will be converted into covalent irreversible probes. Tethering will be extended using the most selective and potent small molecules inhibitors to assess the function of activators such as CREB, and MLL interactions with GACKIX domain of CBP, p300, and ARC105 in cells.